Human immunodeficiency virus type 1 (HIV-1) encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. Tat has multiple domains and consequently the molecular mechanisms by which Tat regulates viral and cellular gene expression are complex. Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of CTD kinases to the HIV-1 promoter. Using an immobilized DNA template assay, we have analysed the effect of Tat on kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that CDK7 (TFIIH) and CDK9 (P-TEFb) both associate with the HIV-1 pre-initiation complex. Hyperphosphorylation of the RNAP II CTD in the HIV-1 pre-initiation complex, in the absence of Tat, takes place at CTD Serine 2 and Serine 5. Analysis of pre-initiation complexes formed in immunodepleted extracts suggest that CDK9 phosphorylates Serine 2 while CDK7 phosphorylates Serine 5. Remarkably, in the presence of Tat, the substrate specificity of CDK9 is altered such that the kinase phosphorylates both Serine 2 and Serine 5. Tat-induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5,6-Dichloro-1-b-D-ribofuranosylbenzimidazole (DRB), a inhibitor of transcription elongation by RNAP II. Analysis of stalled transcription elongation complexes demonstrates that CDK7 is released from the transcription complex between +14 and +36, prior to the synthesis of TAR RNA. In contrast, CDK9 stays associated with the complex through +79. Analysis of CTD phosphorylation indicates a biphasic modification pattern, one in the pre-initiation complex and the other between +36 and +79. The second phase of CTD phosphorylation is Tat-dependent and TAR-dependent. These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex. CDK9 phosphorylation is required for high affinity binding of Tat/P-TEFb to the TAR RNA structure. We have recently shown that CDK9 phosphorylation is uniquely regulated in the HIV-1 preinitiation and elongation complexes. The presence of TFIIH in the HIV-1 preinitiation complex inhibits CDK9 phosphorylation. As TFIIH is released from the elongation complex between +14 and +36, CDK9 phosphorylation is observed. Consistent with these observations, we have demonstrated that purified TFIIH directly inhibits CDK9 autophosphorylation. Using recombinant TFIIH subcomplexes, our results suggest that the XPB subunit of TFIIH is responsible for this inhibition of CDK9 phosphorylation. Interestingly, our results further suggest that the phosphorylated form of CDK9 is the active kinase for RNAP II CTD phosphorylation. The HIV epidemic remains one of the greatest threats to public health, in part because effective vaccines or antiviral agents have been difficult to develop. Although viral reverse transcriptase and protease inhibitors reduce the level of virus replication, the benefit is in most cases short term because of the emergence of resistant viral strains. Tat transactivation is an attractive target for antiviral therapy because Tat is required for viral gene expression not only during exponential growth, but also during activation of the integrated proviral genomes that give rise to drug-resistant strains. Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of cellular transcription elongation factor, P-TEFb to the HIV-1 elongation complex. Tat transactivation is also critically dependent upon the recruitment of cellular transcription factors SPT-5 and Tat-SF1. Using an immobilized DNA template assay, we have analyzed the effect of Tat on P-TEFb kinase activity during the initiation and elongation phases of HIV-1 transcription. Our results demonstrate that Tat induces phosphorylation of Pol II CTD, SPT5 and Tat-SF1 during transcription elongation in a Tat-dependent and TAR-dependent manner. Remarkably, the Tat/TAR-dependent kinase activity of P-TEFb on RNAP II CTD, SPT5 and Tat-SF1 (IC50 1 nM) is ten-fold more sensitive than the Tat-independent kinase activity of P-TEFb (IC50 10 nM) to flavopiridol. The results suggest that the selective inhibition of Tat transactivation by flavopiridol may be due to the sensitivity of the Tat/TAR-dependent kinase activity of P-TEFb on, not one, but three transcription factors which are essential for Tat transactivation. Understanding the biological effect of these post-translational modifications and understanding the mechanism of flavopiridol inhibition will provide important insight into the development of targeted antiviral therapies.